CST Guide:
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Section I: Research Areas<br />
SAPK/JNK Signaling<br />
Growth Factors, UV<br />
RTKs<br />
CrkII<br />
CrkL<br />
Shc<br />
GRB2<br />
HPK1<br />
Cytoplasm<br />
Nucleus<br />
PI3K<br />
14-3-3<br />
SOS<br />
MAPKKKs :<br />
TAK1<br />
Ras<br />
MKK4/7<br />
JNK1/2/3<br />
IRS-1<br />
c-Abl 14-3-3 14-3-3<br />
DUSPs<br />
c-Abl<br />
Cellular Stress,<br />
γ Radiation<br />
Rac<br />
MEKK1/4<br />
MLK2/3<br />
DLK<br />
TpI-2<br />
TAO1/2<br />
NO<br />
Cdc42<br />
Rho<br />
MLKs<br />
POSH<br />
JNK<br />
JNK<br />
FasL, UV<br />
Inflammatory Cytokines<br />
GCK<br />
ASK1<br />
Rac<br />
c-Jun ATF-2 Elk-1 Smad4 p53 NFAT4 NFATc1 Stat3<br />
TRADD<br />
TRAF2<br />
JIP1,2,3<br />
RIP<br />
Daxx<br />
Transcription<br />
Oxidative<br />
Stress<br />
Apoptosis<br />
Cdc42<br />
MAPKKKs<br />
Bax<br />
HSF1<br />
Gα 12/13<br />
14-3-3<br />
Bax<br />
Growth<br />
Differentiation<br />
Survival<br />
Apoptosis<br />
G 12/13 -coupled<br />
Receptors<br />
Gβγ<br />
PI3Kγ<br />
Mitochondrial<br />
Translocation/<br />
Apoptosis<br />
Stress-activated protein kinases (SAPK)/Jun amino-terminal kinases (JNK) are members of the MAPK family and are activated by a variety of environmental stresses, inflammatory<br />
cytokines, growth factors, and GPCR agonists. Stress signals are delivered to this cascade by small GTPases of the Rho family (Rac, Rho, cdc42). As with the other<br />
MAPKs, the membrane proximal kinase is a MAPKKK, typically MEKK1–4, or a member of the mixed lineage kinases (MLK) that phosphorylates and activates MKK4 (SEK) or<br />
MKK7, the SAPK/JNK kinases. Alternatively, MKK4/7 can be activated by a member of the germinal center kinase (GCK) family in a GTPase-independent manner. SAPK/JNK<br />
translocates to the nucleus where it can regulate the activity of multiple transcription factors.<br />
Select Reviews:<br />
Bogoyevitch, M.A., Ngoei, K.R., Zhao, T.T., Yeap, Y.Y., and Ng, D.C. (2010) Biochim. Biophys. Acta. 1804, 463–475. • Chen, F. (2012) Cancer Res. 72, 379–386. • Davies,<br />
C. and Tournier, C. (2012) Biochem. Soc. Trans. 40, 85–89. • Engström, W., Ward, A., and Moorwood, K. (2010) Cell Prolif. 43, 56–66. • Haeusgen, W., Herdegen, T., and<br />
Waetzig, V. (2011) Eur. J. Cell Biol. 90, 536–544. • Verma, G., and Datta, M. (2012) J. Cell Physiol. 227, 1791–1795.<br />
PI3 Kinase/Akt Signaling<br />
Activation of Akt<br />
The serine/threonine kinase Akt/PKB exists as three isoforms in mammals. Akt1 and Akt2 have a wide<br />
tissue distribution, whereas Akt3 is expressed in testes and brain. Akt regulates multiple biological processes<br />
including cell survival, proliferation, growth, and glycogen metabolism. Various growth factors,<br />
hormones, and cytokines activate Akt by binding their cognate receptor tyrosine kinase (RTK), cytokine<br />
receptor, or G protein-coupled receptor (GPCR) and triggering activation of the lipid kinase PI3K, which<br />
generates PIP 3 at the plasma membrane. Akt binds PIP 3 through its pleckstrin homology (PH) domain,<br />
resulting in translocation of Akt to the membrane. Akt is activated through a dual phosphorylation<br />
mechanism. PDK1, which is also brought to the membrane through its PH domain, phosphorylates Akt<br />
within its activation loop at Thr308. A second phosphorylation at Ser473 within the C-terminus is also<br />
required for activity and is carried out by the mTOR-Rictor complex, mTORC2.<br />
Insulin stimulation results in clustering of phospho-Akt at the membrane.<br />
Phospho-Akt (Thr308) (D25E6) XP ® Rabbit<br />
mAb #13038: Confocal IF analysis of C2C12<br />
cells, insulin-treated (100 nM, 15 min; left) or<br />
treated with LY294002 #9901 (50 μM, 2 hr;<br />
right), using #13038 (green). Actin filaments<br />
were labeled with DY-554 phalloidin (red). Blue<br />
pseudocolor = DRAQ5 ® #4084 (fluorescent<br />
DNA dye).<br />
Growth factor stimulation results in phosphorylation<br />
of Akt at Thr308, one of two phosphorylations<br />
necessary for Akt activation.<br />
Phospho-Akt (Thr308) (D25E6) XP ® Rabbit mAb #13038: Western blot analysis of<br />
extracts from NIH/3T3 cells, untreated or treated with Human Platelet-Derived Growth<br />
Factor AA (hPDGF-AA) #8913 (100 ng/ml, 5 min; +), and untreated LNCaP and PC-3<br />
cells, using #1308 (upper) or Akt (pan) (C67E7) Rabbit mAb #4691 (lower).<br />
Lanes<br />
1. NIH/3T3<br />
2. NIH/3T3<br />
3. LNCaP<br />
4. PC-3<br />
kDa<br />
140<br />
100<br />
80<br />
60<br />
50<br />
60<br />
50<br />
1 2 3 4<br />
Phospho-<br />
Akt (Thr308)<br />
Akt (pan)<br />
– + – – hPDGF<br />
Inhibition of Akt Activity<br />
PTEN, a lipid phosphatase that catalyzes the dephosphorylation of PIP 3 , is a major negative regulator of<br />
Akt signaling. Loss of PTEN function has been implicated in many human cancers. Akt activity is also<br />
negatively regulated by the phosphatases PP2A and PHLPP, as well as by the chemical modulators<br />
wortmannin and LY294002, both of which are inhibitors of PI3K.<br />
PTEN expression in human colon<br />
PTEN (D4.3) XP ® Rabbit mAb #9188: IHC analysis<br />
of paraffin-embedded human colon using #9188.<br />
kDa<br />
100<br />
chapter 02: Signaling<br />
Akt-activating treatments<br />
and absence<br />
of PTEN result in<br />
phosphorylation of<br />
Akt at Ser473.<br />
A<br />
80<br />
60<br />
50<br />
80<br />
60<br />
50<br />
PC-3<br />
– +<br />
– +<br />
– –<br />
NIH/3T3<br />
– –<br />
– –<br />
– +<br />
Phospho-Akt (Ser473)<br />
is expressed in human<br />
lung carcinoma.<br />
B<br />
Phospho-<br />
Akt (Ser473)<br />
Akt<br />
wortmannin<br />
LY294002<br />
PDGF<br />
Phospho-Akt (Ser473) (D9E) XP ®<br />
Rabbit mAb #4060: WB analysis of<br />
extracts from PC-3 cells (PTEN null),<br />
untreated or treated with LY294002<br />
#9901/Wortmannin #9951, and NIH/3T3<br />
cells, serum-starved or PDGF-treated<br />
(A), using #4060 (upper) or Akt (pan)<br />
(C67E7) Rabbit mAb #4691 (lower). IHC<br />
analysis of paraffin-embedded human<br />
lung carcinoma (B) using #4060.<br />
© 2002–2015 Cell Signaling Technology, Inc. • We would like to thank Prof. John Blenis, Harvard Medical School, Boston, MA, for reviewing this diagram.<br />
48 For Research Use Only. Not For Use in Diagnostic Procedures. See pages 302 & 303 for Pathway Diagrams, Application, and Reactivity keys.<br />
www.cellsignal.com/cstakt 49